Molecular and biochemical characterization of the transcription factors that mediate the induction of interleukin 2 (IL2) production secondary to co-stimulation by engagement of the CD28 receptor on T-cells has previously identified c-rel and NF-kappa related components as major transcriptional mediators of the response. We have found the CD28 dependent transcriptional stimulation of IL2 occurs through the cooperative interaction of c-rel with CREB/ATF factors and a composite site within the interleukin 2 promoter that contains the previously described CD28RE (CD28 Response Element) and flanking sequences that encompass and non-consensus TPA response element (TRE). This site which is referred to as the CD28/TRE binds cooperatively to a complex containing both c-rel and leucine zipper dimers containing ATF-1 and AFT-4 (CREB-2). DNA binding studies indicate that the CD28/TRE is contacted by a large complex that produces extensive protection (approx. 22 bp) of the cis element. In addition binding and transcriptional activation at the CD28/TRE is highly dependent on the phasing of the regions of the CD28/TRE that bind c-rel and those that bind ATF/CREB, suggesting that protein-protein interaction my mediate the cooperative effects of c-rel and ATF/CREB on transactivation from the CD28/TRE. Complexes contain c-rel and ATF/CREB have been purified from human T-cells stimulated in the presence of antibodies that activate via the CD28 receptor. Biochemical characterization of the purified protein suggest coordinate and cooperative interaction between the components enable the formation of a multi-component complexes that binds tightly to the CD28/TRE to mediate CD28 dependent transcriptional activation.